Impact of tobacco recessive resistance gene va on biological properties of Brazilian Potato virus Y (PVY) isolates

To investigate the role of environmental conditions on the selection of virulent Potato virus Y (PVY) isolates subject to pressure from the recessive resistance gene va in tobacco, a field survey was performed in Brazil where va‐derived genotypes have been recently introduced and now represent less than one‐third of cultivated tobacco genotypes. A serological analysis of 397 leaves collected from different Brazilian tobacco‐growing areas and mainly from plants with symptoms indicated that 52·4% of samples were infected by at least one of the viral species tested. PVY was present in 72·1% of infected samples. The probability of a plant being infected with PVY was reduced in va hosts. However, the biological characterization of PVY isolates on indicator hosts showed that 20 of the 29 tested isolates were able to overcome the alleles of the va gene. Moreover, the observed biological diversity of isolates was higher in susceptible tobacco genotypes than in va‐resistant ones. Comparison of these data with the PVY diversity in French tobacco fields shows that the use of va‐derived genotypes in two environments with contrasting climatic conditions, local hosts and cultural contexts, leads to a similar outcome: the prevalence of virulent isolates. These results strongly suggest an important role of the va gene in the modification of PVY populations.     

Effect of passage of a Potato virus Y isolate on a line of tobacco containing the recessive resistance gene va2 on the development of isolates capable of overcoming alleles 0 and 2

Control strategies developed for plant viral diseases through breeding programs can be impaired by adaptive response of pathogens. A few years after the deployment in France of improved tobacco genotypes introgressed with alleles of the Potato virus Y (PVY) recessive resistance gene va, necrotic symptoms associated with PVY infection have been reported on these cultivars. Due to the lack of efficient alternative methods to control PVY, the va resistance sources have to be managed according to viral parameters, such as the dynamic of emergence of virulent variants. The effects of va alleles on the evolution of PVY was tested in experimental conditions using a PVY infectious clone and two couples of resistant tobacco near isogenic lines BVA/Bva ⁰ and FVA/Fva ² both allelic for the va gene. Infection efficiencies data showed that a single passage on Fva ² line led to the selection of virulent viral populations able to overcome both va ⁰ and va ² alleles. Sequence analyses of va ² derived PVY variants revealed that the acquisition of the capacity to overcome va ² resistance is associated with single point mutations at two different nucleotide positions in the central part of the VPg. The described PVY adaptation process to overcome resistance mediated by different va alleles should be considered for the future development of durable and integrated strategies of management of PVY infections in tobacco crops.     

A novel type of Potato virus Y recombinant genome,determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVY^NTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVY^NTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVY^NTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVY^E strain. To distinguish PVY‐AGA and PVY‐MON from other PVY^NTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVY^N‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY^N‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.     

Brazilian Potato virus Y isolates identified as members of a new clade facilitate the reconstruction of evolutionary traits within this species

Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life‐history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y^U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y^U isolates made it possible (i) to identify specific amino acid residues in the N‐terminal of the CP protein and (ii) to assign some Y^U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY ‘Brazilian’ clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time.     

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVY^O (pathotype and serotype O), PVY^NW (pathotype N and serotype O), and PVY^NTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVY^NTN isolates, and the lowest ones with PVY^O isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.     

Resistance in Australian barley (Hordeum vulgare) germplasm to the exotic pathogen Puccinia striiformis f. sp. hordei,causal agent of stripe rust

Eighty‐eight Australian and 10 international barley cultivars were assessed for resistance to the barley stripe (yellow) rust pathogen, Puccinia striiformis f. sp. hordei (Psh). All cultivars were tested for seedling resistance to two UK‐derived isolates of Psh (11.01 and 83.39) that were shown to differ in virulence based on responses on 16 differential barley genotypes. The 98 barley cultivars differed substantially in stripe rust response; 45% were susceptible to Psh 11.01, 53% to Psh 83.39 and 44% to both isolates. The observed diverse infection types (ITs) suggest the presence of both known and uncharacterized resistance. However, further multipathotype tests are required for accurate gene postulation. The Yerong × Franklin (Y×F) doubled haploid (DH) population was phenotypically assessed as seedlings using both Psh isolates. Yerong and Franklin were immune and highly resistant, respectively, to both isolates used in this study. Marker‐trait and QTL mapping identified a major effect on the long arm of chromosome 7H contributed by Franklin in response to all isolates. The resistance of Yerong was mapped to 113·96 and 169·38 cM on chromosome 5HL in response to Psh 11.01 and 83.39, respectively. The Psh resistance sources identified in this study can be used for further genetic analysis and introgression for varietal improvement.     

Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia

Resistance of Brassica napus (oilseed rape, canola) conferred by three different major resistance genes has been overcome by changes in virulence of Leptosphaeria maculans populations in France and Australia. In South Australia where B. napus cultivars with major gene resistance derived from Brassica rapa ssp. sylvestris were grown extensively, resistance was rendered ineffective within 3 years of commercial release of the cultivar. Disease severity was higher on cultivars with sylvestris-derived resistance than cultivars with polygenic resistance. This Australian situation is compared to that in France, where resistance conferred by the Rlm1 gene was overcome nation-wide in 5 years under commercial cropping practices, and also where a source of resistance introgressed into B. napus from B. juncea was rendered inefficient in 3 years in experimental field plots near Rennes.     

Durability of natural and transgenic resistances in rice to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis>

A monogenic recessive resistance to Rice yellow mottle virus (RYMV) found in the Oryza sativa indica cultivar Gigante and in a few Oryza glaberrima cultivars provided a higher level of resistance than either a polygenic partial resistance found in some japonica cultivars which delayed symptom expression or transgenic resistances which were partial and temporary. This high resistance was overcome by several isolates, but the percentage of such virulent isolates in the fields was low. There was no relationship between the virulence of an isolate towards the high resistance and its aggressiveness in other cultivars. Isolates with either of the two components of pathogenicity – virulence and aggressiveness – were found in each strain and in all regions of Africa, in both wild and cultivated grass species. There was no loss of fitness of resistance-breaking (RB) isolates as they were not counter-selected, impaired or outperformed after serial passages in susceptible cultivars, even in mixture with avirulent quasi-isogenic wild type isolates. Resistance breaking was highly dependent on the amount of virus inoculated and on the mode of transmission. Implications of these results for the durability of the resistances to RYMV and for the development of integrated disease management strategies are discussed.     

Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.     

Pathotypic and genetic diversity in the population of Rhizoctonia solani AG1‐IA causing rice sheath blight in China

Sheath blight, caused by Rhizoctonia solani AG1‐IA, is one of the most serious diseases of rice. In this study, a total of 175 isolates of R. solani AG1‐IA were collected from five rice‐growing regions in China. Pathogenicity tests revealed that all isolates were virulent to five cultivars with different levels of resistance at the rice seedling stage in the greenhouse. There was considerable variation in aggressiveness, and the isolates were classified into three pathotypes based on disease severity, with moderately virulent isolates prevalent in the population. Forty‐three haplotypes were identified based on ITS sequencing, and 39 haplotypes were distinct among isolates. There were high levels of haplotype diversity and nucleotide diversity within the populations of R. solani AG1‐IA. High gene flow (Nm = 1·63–5·22) was detected, consistent with relatively low differentiation between pairs of populations. Five populations were divided into two distinct clusters by the unweighted pair group method with arithmetic mean (UPGMA), and no spatial population differentiation was discernible. The majority (97·8%) of genetic diversity was distributed among isolates within populations, with only 2·2% of the genetic diversity attributed to differences among populations. The star‐like shape of the haplotype network provided evidence of signatures of population expansion in recent history. No significant relationships were found between the genetic diversity and aggressiveness or geographic origin among populations of R. solani AG1‐IA. These results highlight that the population characteristics of R. solani AG1‐IA should be taken into account in evaluating the germplasm resistance of rice cultivars to sheath blight.     

Genetics of virulence in Ascochyta rabiei

In order to critically test the hypothesis that virulence variation in the Ascochyta rabiei/chickpea pathosystem is a discrete character under simple genetic control, a genetic cross was made between a highly virulent isolate of A. rabiei from Syria and a less virulent isolate from the USA. Two independent virulence assays conducted by inoculating susceptible and resistant chickpea cultivars under controlled conditions with 77 independent progeny isolates from this cross revealed a continuous distribution of disease phenotypes. Bimodality, as would be predicted for the segregation of virulence under simple genetic control, was not supported by statistical tests of the progeny phenotype distribution. anova revealed highly significant pathogen‐genotype × host‐genotype interactions demonstrating the segregation of genes controlling specialization on the two cultivars tested. These interactions could be localized to two isolates that changed virulence rank on the cultivars. It was concluded that variation in virulence to these two cultivars is under quantitative genetic control. If this conclusion applies to other cultivars, it can be speculated that the discrete categories of virulence variation identified in previous studies were probably the result of incomplete sampling of host resistance or pathogen virulence variation and/or of selection for increased virulence in contemporary A. rabiei populations.     

A classification of <Emphasis Type="Italic">Pepper yellow mosaic virus</Emphasis> isolates into pathotypes

Pepper yellow mosaic virus (PepYMV) is the most important potyvirus infecting sweet pepper in Brazil. In this study, twenty isolates of PepYMV were obtained from commercial sweet pepper crops. To confirm virus identity, the coat protein gene was completely sequenced for eleven of these isolates, and partially sequenced for the other nine isolates. The amino acid identities obtained were above 93% when compared with the sequence of a characterized PepYMV isolate (AF348610). Extracts of Nicotiana tabacum cv. TNN plants infected with the different isolates were used to inoculate the differential series of Capsicum spp cultivars containing the genes pvr2 ¹ , pvr2 ² , pvr2 ³ , pvr2 ⁴ , and Pvr4. Using the same criteria established for Potato virus Y (PVY), fourteen isolates of PepYMV could be classified as known pathotypes described for PVY, that is: 1.2 (2 isolates), 1.3 (6) and 1.2.3 (6). The remaining six isolates, 1.3 (2) and 1.2.3 (4) could not be classified into the typical pathotypes of PVY because they were also virulent on Serrano Criollo de Morellos—334 (C.M 334) which carries the pvr2 ³ and Pvr4 genes. To classify the PepYMV into pathotypes and counter the biological diversity found in this species we propose the utilization of 2^x for the ability to overcome the correspondent allele of the pvr2 locus and 4 for the capacity to break down the Pvr4 gene. Using this criterion we could classify the PepYMV into five pathotypes: 2¹.2²; 2¹.2³; 2¹.2².2³; 2¹.2³. 4 and 2¹.2².2³. 4.     

Diaporthe sclerotioides exhibits no host specificity among cucurbit species

Plant–fungal specificity between cucurbitaceous crops and Diaporthe sclerotioides, the causal agent of black root rot, was studied using cucumbers (Cucumis sativa), melons (Cucumis melo), pumpkins (Cucurbita maxima), watermelons (Citrullus lanatus) and bottlegourd (Lagenaria siceraria var. gourda). Twelve D. sclerotioides isolates from these cucurbit species were cross‐inoculated. The virulence of the isolates was evaluated as the area under the disease progress curve (AUDPC). All cucurbit species were susceptible to each isolate, but AUDPCs were significantly different among the hosts, with the order of greatest to least being melon, cucumber, watermelon, bottlegourd and finally, pumpkin. The infectiveness of isolates was assessed as the quantity of D. sclerotioides DNA detected in the hypocotyls of seedlings 2 weeks after inoculation using a real‐time PCR protocol. The fungal DNA quantities varied among the species in the same order as the AUDPCs. Whilst there were statistically significant correlations between the virulence and infectiveness of D. sclerotioides isolates in cucumbers, melons and bottlegourds, their coefficients of determination were not high (r² < 0·6). Orthogonal contrasts indicated no specificity in either the fungal virulence or infectiveness between D. sclerotioides isolates and the cucurbit hosts from which these isolates originated. Thus, although the degree of host susceptibility to D. sclerotioides varies among cucurbit species, the absence of specificity to the host species in either virulence or infectiveness suggests the pathogen may spread via various cucurbit crops, irrespective of their original host species.     

Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe

This paper describes the first large-scale Europe-wide survey of avirulence alleles and races of Leptosphaeria maculans. Isolates were collected from the spring rape cultivar Drakkar, with no known genes for resistance against L. maculans, at six experimental sites across the main oilseed rape growing regions of Europe, including the UK, Germany, Sweden and Poland. Additionally in Poland isolates were collected from cv. Darmor, which has resistance gene, Rlm9. In total, 603 isolates were collected during autumn in 2002 (287 isolates from Germany and the UK) and 2003 (316 isolates from Poland and Sweden). The identity of alleles at eight avirulence loci was determined for these isolates. No isolates had the virulence allele avrLm6 and three virulence alleles (avrLm2, avrLm3 and avrLm9) were present in all isolates. The isolates were polymorphic for AvrLm1, AvrLm4, AvrLm5 and AvrLm7 alleles, with virulence alleles at AvrLm1 and AvrLm4 loci and avirulence alleles at AvrLm7 and AvrLm5 loci predominant in populations. Virulent avrLm7 isolates were found at only one site in Sweden. Approximately 90% of all isolates belonged to one of two races (combinations of avirulence alleles), Av5-6-7 (77% of isolates) or Av6-7 (12%). Eight races were identified, with four races at frequencies less than 1%. The study suggested that Rlm6 and Rlm7 are still effective sources of resistance against L. maculans in oilseed rape in Europe. The results are comparable to those of a similar survey done in France in autumn 2000 and 2001.     

Identification of isolate‐specific resistance QTLs to phytophthora root rot using an intraspecific recombinant inbred line population of pepper (Capsicum annuum)

Quantitative trait loci (QTL) for resistance to phytophthora root rot caused by Phytophthora capsici were investigated using two Korean P. capsici isolates and 126 F₈ recombinant inbred lines derived from a cross of Capsicum annuum line YCM334 (resistant parent) and local cv. Tean (susceptible parent). The experimental design was a split plot with two replications. Highly significant effects of pathogen isolate, plant genotype, and genotype × isolate were detected. QTL mapping was performed using a genetic linkage map covering 1486·6 cM of the pepper genome, and consisted of 249 markers including 136 AFLPs (Amplified Fragment Length Polymorphisms), 112 SSRs (Simple Sequence Repeats) and one CAPS (Cleaved Amplified Polymorphic Sequence). Fifteen QTLs were detected on chromosomes 5 (P5), 10 (P10), 11 (P11), Pb and Pc using two data processing methods: percentage of wilted plants (PWP) and relative area under the disease progress curves (RAUDPC). The phenotypic variation explained by each QTL (R²) ranged from 6·0% to 48·2%. Seven QTLs were common to resistance for the two isolates on chromosome 5 (P5); six were isolate‐specific for isolate 09‐051 on chromosomes 10 (P10) and Pc, and two for isolate 07‐127 on chromosomes 11 (P11) and Pb. The QTLs in common with the major effect on the resistance for two isolates explained 20·0–48·2% of phenotypic variation. The isolate‐specific QTLs explained 6·0–17·4% of phenotypic variation. The result confirms a gene‐for‐gene relationship between C. annuum and P. capsici for root rot resistance.     

Development of a polyvalent RT‐PCR detection assay covering the genetic diversity of Cherry capillovirus A

The variability of Cherry capillovirus A (CVA) was analysed using a short, 275‐bp region of the viral RNA‐dependent RNA polymerase gene amplified by a polyvalent RT‐PCR assay. As for other members of the family Betaflexiviridae, CVA appears to show significant diversity, with an average pairwise nucleotide divergence of 9·4% between isolates in the analysed region. Phylogenetic analyses provide evidence for the existence of at least five clusters of CVA isolates, one of which is associated with noncherry hosts of the virus, providing evidence that transmission of CVA isolates between cherry and noncherry hosts is probably rare. Comparison of existing detection techniques using a panel of CVA isolates representative of the various phylogenetic groups indicated that dot‐blot hybridization assays show high polyvalence but may lack the sensitivity to detect CVA in some samples. On the other hand, available detection primers failed to amplify a wide range of CVA isolates. Partial genome sequencing of two divergent isolates allowed the identification of conserved genomic regions and the design of new primer pairs with improved polyvalence. These new primer pairs were used to develop PCR assays allowing the reliable detection of CVA isolates belonging to all phylogenetic clusters.     

Effects of isolate virulence and host susceptibility on development of early blight (Alternaria solani) on tomato*

Early blight of tomato (Lycopersicon esculentum) caused by Alternaria solani has the potential to become one of the most serious diseases throughout the tomato‐producing regions of Greece. Controlled environment experiments were conducted to study the virulence of A. solani isolates and the susceptibility of commercial tomato cultivars and hybrids to early blight. The isolates used, derived from naturally infected tomato plants during the period 1997/1998, differed significantly (P>0.05) in the rate of mycelial growth as well as in their ability to sporulate in vitro. No correlation (R²= 0.33) was found between mycelial growth and conidia production. Isolates of A. solani were virulent to young tomato plants (cv. Ace 55VF), although they differed significantly (P>0.05) in the intensity of symptoms produced on leaves, stems, petioles and flowers. Defoliation was linearly related (R²= 0.87) to the percentage of leaf area with symptoms. Twenty‐three tomato cvs. or F₁ hybrids were evaluated for their susceptibility to early blight. The cultivars or hybrids were arbitrarily categorized as immune, highly tolerant, tolerant, moderately tolerant, susceptible and highly susceptible based on a percent disease index range: 0%, 1‐9%, 10‐24%, 25‐49%, 50‐74% and 75% or more respectively. None of the cultivars or hybrids tested was immune or tolerant to A. solani infection.